Citation
- Authors: Feng J. et al.
- Year: 2022
- Journal: Nat Commun 13 4064
- Applications: in vitro / DNA / FectoPRO
- Cell type: Expi293F
Description: Human embryonic kidney Fibroblast
Known as: Expi 293-F, Expi, HEK-293 Expi
Method
0.8 mg plasmid was incubated with 0.8 mL FectoPRO (Polyplus) in Opti-MEM (Gibco) for 10 min at room temperature and then added to 1 L of Expi293 cells cultured in Expi293 medium (Thermo Fisher Scientific). 24 hrs after transfection, valproic acid and glucose were added to the culture to final concentrations of 3 mM and 0.4% w/v, respectively. Six days after transfection, secreted proteins were purified from the media by His-tag affinity chromatography.
Abstract
Here, we study the gamete fusogen HAP2 from Cyanidioschyzon merolae (Cyani), an extremophile red algae that grows at acidic pH at 45 °C. HAP2 has a trimeric postfusion structure with similarity to viral class II fusion proteins, but its prefusion structure has been elusive. The crystal structure of a monomeric prefusion state of Cyani HAP2 shows it is highly extended with three domains in the order D2, D1, and D3. Three hydrophobic fusion loops at the tip of D2 are each required for postfusion state formation. We followed by negative stain electron microscopy steps in the process of detergent micelle-stimulated postfusion state formation. In an intermediate state, two or three linear HAP2 monomers associate at the end of D2 bearing its fusion loops. Subsequently, D2 and D1 line the core of a trimer and D3 folds back over the exterior of D1 and D2. D3 is not required for formation of intermediate or postfusion-like states.